Nucleic Acid Nanocapsules for Drug Delivery and Targeted Gene Knockdown

ABSTRACT

The present disclosure provides multifunctional nanoparticles. More particularly, the present disclosure relates to multifunctional nanoparticles having one or more of nucleic acid ligands; and methods of using such nanoparticles for treatment and/or diagnosis of diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/016,511, filed Jun. 22, 2018, which claims the benefit ofU.S. Provisional Patent Application No. 62/523,929, filed Jun. 23, 2017,both of which are incorporated herein by reference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The Sequence Listing iscontained in the file created on Oct. 29, 2020, having the file name“17-728-US-DIV_Sequence-Listing_ST25.txt” and is 4 kilobytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure provides multifunctional nanoparticles. Moreparticularly, the present disclosure relates to multifunctionalnanoparticles having one or more of nucleic acid ligands; and methods ofusing such nanoparticles for treatment and/or diagnosis of diseases andconditions.

Description of the Related Art

Nucleic acid-based therapeutics have become increasingly important drugcandidates in recent years thanks to the advent of nanoparticle drugcarriers. Such nucleic acids, including siRNA, antisense DNA, andcatalytic nucleic acids have been shown to be effective tools forinitiating intracellular gene regulation. However, these moleculessuffer in their overall efficacy due to their inherent chemicalinstability. Recent studies have shown that the tight packing of nucleicacids at the surface of a nanoparticle can result in advantageouscellular delivery properties that the nucleic acid sequence alone cannotachieve. Such structures, referred to as spherical nucleic acids (SNAs)provide desirable delivery properties including increased cellularuptake through endocytosis and the prolonged half-life of nucleic acids.The later property is particularly important for the delivery of nucleicacids that rely on their folded structure to impart therapeutic effectsincluding DNAzymes, ribozymes and aptamers.

Using such an SNA configuration on a colloidal gold nanoparticlescaffold, it was recently shown that a functional ribozyme could besuccessfully delivered into cells for regulating gene expression.However, as these structures were attached to inorganic nanoparticles(NPs), much of the particle core could not contribute to the overalltherapeutic function other than to provide a scaffold on which to buildthe SNA configuration.

Therefore, there remains a need for assembling an SNA-like structure atthe surface of a nanomaterial that could be utilized as a drug carrierand as a scaffold for further RNA and DNA functionalization. Althoughthere are numerous soft material based approaches to drug encapsulation,the specific challenge remains to develop a material that can be rapidlyfunctionalized with nucleic acids and rapidly release a drug cargo. Theparticle could then impart enhanced uptake due to the properties of theSNA, coupled with gene knockdown potential and small molecule drugdelivery in a single construct.

SUMMARY OF THE DISCLOSURE

One aspect of the disclosure provides multifunctional nanoparticlesincluding one or more of nucleic acid ligands covalently attached to aparticle including non-polymeric amphiphiles,

-   -   wherein hydrophobic groups of the amphiphiles are arranged        toward the particle interior, and    -   wherein hydrophilic groups of the amphiphiles are at the        particle surface and are crosslinked through a triazole,        thioether, or alkenyl sulfide group with one or more linkers        cleavable by one or more intracellular or extracellular release        agents.

Another aspect of the disclosure provides conjugates including themultifunctional nanoparticle of the disclosure and at least onetherapeutic agent or diagnostic agent, wherein the multifunctionalnanoparticle encapsulates the therapeutic agent.

Also disclosed herein are pharmaceutical compositions of themultifunctional nanoparticles of the disclosure or the conjugates of thedisclosure. Examples of such compositions include those having at leastone pharmaceutically acceptable carrier, diluent, and/or excipienttogether with a multifunctional nanoparticle or a conjugate as describedherein.

Another aspect of the disclosure provides methods of treating a diseaseor disorder, including administering to a subject in need thereof aneffective amount of the conjugate of the disclosure, wherein the linkeris cleavable by one or more intracellular or extracellular release agentpresent in the subject, thus releasing the therapeutic agent ordiagnostic agent. For example, in some embodiments, the disease ordisorder is cancer, infection (e.g., bacterial, viral, or parasitic),pain, asthma, inflammation, neurological disease or disorder (e.g.,Alzheimer's disease, Parkinson's disease, etc.). In certain embodiments,the disease or disorder is asthma, inflammation (e.g., asthma-inducedinflammation or chronic obstructive pulmonary disease (COPD)-inducedinflammation), infection (e.g., lower respiratory infections), orcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the methods and compositions of the disclosure, and areincorporated in and constitute a part of this specification. Thedrawings illustrate one or more embodiment(s) of the disclosure, andtogether with the description serve to explain the principles andoperation of the disclosure.

FIG. 1 shows a Stepwise assembly of nucleic acid nanocapsules. Thetrialkyl-modified surfactant 1 shown at top left is placed in water andquickly self-assembles into a micelle structure presenting alkynes atits surface. An esterified diazido cross-linker 2 is used to stabilizethe structure using copper I catalyzed click chemistry. Remainingalkynes are used as a point of attachment for thiolated DNA moleculesthrough a photodriven cross-linking step. After assembly of the NANstructure it can be further functionalized using an enzyme-mediatedassembly approach with T4 DNA ligase to introduce DNAzymes to the NAN'ssurface.

FIG. 2 illustrates characterization of NANs of Example 1 pre- andpost-DNA assembly. (A) Dynamic light scattering measurements ofparticles prior to and after DNA conjugation, cross-linked micelle (CM)and NAN. (B) TEM micrograph showing the average size of uranyl acetatestained NANs.

FIG. 3 illustrates characterization of NANs of Example 1 pre- andpost-DNA assembly. (A) Zeta potential measurements of the nanocapsulespre and post functionalization with DNA. (B) 1% agarose gel showingmovement of fluorescent NANs loaded with rhodamine B through an electricfield post DNA functionalization (1, cross-linked micelle prior to DNAattachment; 2, NAN, post DNA attachment).

FIG. 4 illustrates enzyme-mediated release of internal cargo of NANs ofExample 1. (A) Total emission in a sample of NANs cross-linked withcompound 2 containing rhodamine B, post treatment with esterase overtime in buffer. (B) Representative plots of ester-linked NANs andnonester-linked NANs (made with non-esterified crosslinker 3) versustime after 2 h of treatment with esterase. (C,D) Evaluation of celltoxicity in HeLa cells in the presence of NANs loaded with camptothecin.Results indicate a dose dependent decrease in cell viability post 24 hincubation with ester-linked NANs (dark gray bars) in (C), and a limitedeffect from NANs linked by a non-esterified linker (black bars) in (D).Concentrations indicate total camptothecin loaded within NANs. Controlis 50 μM free drug. These results indicate that the release of theapoptotic drug is dependent upon the presence of both the esterifiedcross-linker and the esterase. (E) Cell viability tests of HeLa Cellsincubated with increasing concentrations of NANs.

FIG. 5 shows cleavage of mRNA truncates using DNAzyme-functionalizedNANs and cellular uptake. (A) Polyacrylamide gel electrophoresis showingthe cleavage of RNA induced by DNAzyme functionalized NANs after 4 h at37° C.: lane 1, truncated mRNA free in solution; lane 2, post incubationwith DNAzyme-NANs; lane 3, mutated DNAzyme-NANs without salts; lane 4,mutated DNAzyme-NANs with salts; and lane 5, DNAzyme off particle,showing complete cleavage of the truncated mRNA target. Mutated DNAzymedetails in Table 1. Panel B shows the DNAzyme-NAN is able to cleave afluorescently labeled mRNA truncate that is quenched prior to cleavagebut becomes fluorescent post cleavage. In the presence of theDNAzyme-functionalized NANs, the mRNA truncate (10 nM) is rapidlycleaved as shown above. DNAzyme concentrations used in the ligationreaction can result in multiple DNAzymes per NAN. Fluorescence tracesare shown to indicate activity versus lack of activity. Free DNAzyme(500 nM) along with NANs functionalized with mutated DNAzyme-NANs (bothat 100 nM) are shown as positive and negative controls for activity,respectively. (C-F) Cells treated with 1 μM rhodamine B loaded NANs: (C)green emission channel indicating the location of the rhodamine B NANsin cells; (D) Lysotracker Red staining of lysosomes and endosomes withincells; (E) co-localization of NANs and lysosomes showing the NANs enterthe cells through endocytosis; (F) brightfield image and overlay. Scalebar is 25 μm.

FIG. 6 shows a stepwise assembly of peptide-crosslinked Nucleic AcidNanocapsules (pep-NANs).

FIG. 7 shows a stepwise assembly of gold nanoparticle encapsulatedpeptide-crosslinked Nucleic Acid Nanocapsules (pep-Au-NANs).

FIG. 8 illustrates assembly and programmed degradation of peptidecrosslinked nucleic acid nanocapsules (pep-NANs). Peptide sequences ofinterest are modified with terminal cysteines to provide thiolatedattachment points for covalently photo-cross-linking peptides to thealkyne-modified nanocapsule surface. The cross-linkers are incorporatedusing thiol-yne chemistry in the presence of a photoinitiator and UVlight.

FIG. 9 shows nanoscale characterization of peptide cross-linked NANs.(A) Representative DLS measurement of peptide-cross-linked NANs (DLS andzeta potential values are in Table 2). (B) Representative transmissionelectron micrographs showing peptide cross-linked NANs stained with 0.5%uranyl acetate for both CathB and MMP9 peptide substrates. Dottedoutline indicates region of micrograph expanded for CathB NANs. (C) 1%agarose gel showing the change in mobility of individual pep-NANs(CathB-NAN and MMP9-NAN) post attachment of a thiolated polyT20 DNAligand using UV light-driven thiol-yne chemistry.

FIG. 10 illustrates fluorescence monitoring of pep-NAN degradation. (A)Representative raw fluorescence intensity plots showing the release ofdye from MMP9-NANs cross-linked with MMP9 peptide in the presence ofMMP9 enzyme. One μM MMP9-NANs were treated with 0.5 μg of MMP9 andmonitored for 3 h. (B) Dye-loaded CathB-NANs in the presence of 0.5 μgof cathepsin B monitored for 3 h. (C) Fluorescence monitoring ofcathepsin B activity on MMP9-NANs and the effect of varying pH on MMP9cleavage rates. Exposure of MMP9-NANs to cathepsin B and MMP9 at pH 5resulted in no observable change in fluorescence. (D) Fluorescence plotsof MMP9-NANs treated with MMP1 and MMP2 lacked the specificity to cleavethe MMP9-NANs at pH 7. All experiments were conducted in triplicate.

FIG. 11 shows confocal and electron microscopy of pep-NANs. (A) Confocalmicroscopy of pep-NANs (MMP9 and CathB) indicating cellular uptake. (B)Schematic depicting the assembly of a pep-Au-NAN where the cross-linkerconsists of either esters, diols, or peptides, cross-linked using eitherCu(I) azide alkyne catalyzed click chemistry or thiol-yne chemistry,depending on the terminal modification presented by the cross-linker.(C) TEM of diazido-diol (compound 3) cross-linked NANs, individualshells could be seen surrounding the Au NP by TEM when stained with 0.5%uranyl acetate. (D) TEM of MMP9-NANs formed in the presence of analkane-modified AuNP. Similar to the diol-cross-linked NANs, individualparticles can be seen containing AuNPs incorporated into their centers.

FIG. 12 shows cellular uptake of AuNP embedded NANs visualized throughcell sectioning and staining using TEM. (A) TEM of a diazido-diolcross-linked NAN inside a sectioned HeLa cell. (B) Ester-Au-NANs, usedas a positive control, observed within endosomes by TEM. In TEMmicrograph of (C) CathB-Au-NANs and (D) MMP9-Au-NANs intact peptideshells can be observed within the cells. (E) HeLa cells treated with PMA(MMP9 inducer) for 21 h show no toxicity up to 150 ng/mL. (F) HeLa cellstreated with PMA for 21 h followed by incubation with 40 μM MMP9-NANsloaded with 2.5% camptothecin. The results indicate that only after PMAinduction of MMP9 is toxicity of the drug loaded pep-NANs observed.

FIG. 13 illustrates characterization and stability studies of theDNAzyme NAN structure of Example 5. (A) Dynamic light scattering datapre- and post-DNAzyme ligation to the nanocapsule. (B) Schematicdepicting the cleavage of the ester-linked, dye-labeled nanocapsule,resulting in the release of fluorescent DNAzymes bound to surfactantmolecules. (C) 3% agarose gel showing the shift in free DNAzyme vs.DNz-NAN vs. the enzymatic cleavage products after 4 hours of esterasetreatment. (D) PCR amplification of full length DNAzyme after incubationof free DNAzyme and DNAzyme-NANs in PBS or 10% FBS in PBS. Dnz-Extrepresents the extension of the DNAzyme on the 5′ end due to the primerannealed to the DNA anchor at the particles surface, indicating fulllength DNAzyme remains intact post exposure to serum nucleases.

FIG. 14 illustrates fluorometric kinetic analysis of mRNA cleavage ratesfor a free DNAzyme versus a DNAzyme-NAN. (A) Initial rates of cleavagefor the free DNAzyme with various concentrations of mRNA substrate (B)Initial rates of cleavage for the immobilized DNAzymes ligated toDNA-surfactants on the NANs. For both (A) and (B), each data point isthe average of triplicate runs, with standard deviations reported. (C)Maximal rates of cleavage are plotted as a function of mRNA substrateconcentration. The free DNAzyme (triangles) trace is plotted versusimmobilized DNAzymes tethered to DNA-surfactant molecules (diamonds).The plots indicate similar rates of cleavage but that the overallcatalytic activity of the DNAzyme is slightly more efficient than theimmobilized DNz-NAN, interpreted as due to steric effects at thenanocapsules surface due to DNAzyme crowding.

FIG. 15 illustrates cellular uptake and targeted gene knockdown byDNAzyme-NANs in MCF-7 cells. (A) Confocal microscopy of free DNAzymeslabeled with TYE-665 dye transfected into MCF-7 cells and monitored forevidence of cellular uptake post 4 hours of incubation. (B) Confocalmicroscopy of DNz-NANs incubated with cells without transfection agentsin which the DNAzyme on the NAN was also labeled with TYE-665 dye. Inboth cases, TYE signal could be observed in the perinuclear region ofthe cells. All scale bars are 20 μm. (C) GATA-3 mRNA knockdown in MCF-7cells. 250 nM DNz-NAN is capable of knocking down mRNA by 60% comparedto untreated cells (N=3, p=0.0016). There is no significant differencein mRNA knockdown with 150 nM siRNA and 250 nM NAN (N=3, p=0.406). Errorbars represent the standard error of the mean (N=3). (D) MCF-7 cellstreated with 250 nM DNz-NANs, monitored for GATA-3 mRNA levels viaqRT-PCR at times ranging from 4 to 48 hours of incubation.

FIG. 16 illustrates mRNA cleavage by DNAzyme-surfactants onAuNP-templated lipid bilayers. (A) Assembly components of the lipidbilayer templated on a gold nanoparticle (Au NP), including thiolatedDNA (purple) which is adsorbed on to the surface of the Au NP (dark graycircle) alongside a thiolated PEG DSPE lipid (gray). A fluorescentlylabeled mRNA truncate (purple) is hybridized to the thiolated DNA anchorusing a complementary DNA bridge (light green). (B) The assembledconstruct shields the mRNA target from the DNz-surfactants. (C) Theassembled construct is roughly 40 nm in size as shown by TEM, scale bar50 nm. (D) TEM image showing the lipid bilayers assembled on Au NPs,scale bar 100 nm. (E) Fluorescence from the mRNA truncate within the AuNP lipoplex post incubation with either the DNAzyme, DNAzyme surfactant,or a salt solution control. Data reported is the average of three trialswith accompanying standard deviation.

FIG. 17 illustrates HeLa cells viability when treated with unloaded NANsof Example 1 and NANs of Example 1 loaded with ABT-737 (a small moleculeinhibitor of Bcl-2 family proteins).

DETAILED DESCRIPTION OF THE INVENTION

Before the disclosed processes and materials are described, it is to beunderstood that the aspects described herein are not limited to specificembodiments, or examples, and as such can, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular aspects only and, unless specifically definedherein, is not intended to be limiting.

In view of the present disclosure, the methods and compositionsdescribed herein can be configured by the person of ordinary skill inthe art to meet the desired need. In general, the disclosed materialsand methods provide improvements in multifunctional nanoparticles (alsoreferred herein as a nucleic acid nanocapsules or NANs or nanocapsules).For example, the multifunctional nanoparticles of the disclosure asdescribed herein are capable in encapsulating small molecule drugs ordyes or rapidly functionalized with therapeutic nucleic acid ligands(such as a DNAzyme or siRNA sequence). In addition, the multifunctionalnanoparticles of the disclosure as described herein are capable ofnucleic acid delivery and targeted gene knockdown. Unexpectedly, incertain embodiments, the multifunctional nanoparticles can degrade inthe presence of release agents commonly found within a cell (e.g.,peptidase, protease, or esterase), therefore enhancing its degradationafter deployment in an enzyme-specific fashion. The benefits of themultifunctional nanoparticles of the disclosure as described hereinaddress a number of current and important drug delivery hurdles presentin the art, such as the ability to easily functionalize the surface of adrug delivery vehicle for therapeutic or targeting applications,biodegradability, and the capacity for combination therapy, as theinterior can be loaded with one drug, and the surface modified with aseparate therapeutic biomolecule. In certain embodiments, themultifunctional nanoparticle of the disclosure as described herein, areparticularly applicable to the delivery of hydrophobic small moleculedrugs in conjunction with therapeutic oligonucleotides (siRNA, antisenseoligonucleotides, microRNA, aptamers, DNAzymes, Ribozymes, etc.) thatare useful for intracellular gene knockdown and altering proteinexpression levels. For example, the multifunctional nanoparticles of thedisclosure as described herein deliver camptothecin, a topoisomeraseinhibitor and cancer drug, along with a DNAzyme specifically designed totarget the cleavage of GATA-3 mRNA. GATA-3 is a transcription factorthat plays an important role in inflammation pathways by initiatingdownstream TH1 and TH2 cell differentiation. Cleavage of mRNA can resultin blocked protein translation. If the DNAzyme on the nanoparticle isdesigned to target and cleave the mRNA transcript that encodes forGATA-3, it could prevent its downstream expression and thus prevent theimportant upstream steps involved in inflammation responses. Here, themultifunctional nanoparticle of the disclosure conjugated to DNAzymeenabled cellular uptake of the DNAzyme and resulted in specific andpersistent knockdown of a target gene (e.g., 60%) for several hours. TheDNAzyme exhibited this activity without the use of traditional cationictransfection agents and further chemical modifications.

In certain embodiments, the multifunctional nanoparticles of thedisclosure as described herein are modular in nature, which makes theman excellent drug delivery vehicle for a number of different drug cargosand drug-biomolecule combination therapies. The modular nature of thenanoparticles of the disclosure as described herein allows a secondtherapeutic ligand, such as a nucleic acid ligand that, for example, canelicit protein knockdown, to be covalently attached to the surface ofthe particle. The ease of mixing and matching cargo and nucleic acidligands makes the nanoparticles attractive along with itsbiodegradability and nontoxic components.

The multifunctional nanoparticles of the disclosure as described herein,for example, may offer several advantages, such as the ability to mixand match the cleavable linkers and the nucleic acid linkers and/ordiscrete population size (e.g. about 20 nm in size). In certainembodiments, the multifunctional nanoparticles of the disclosure asdescribed herein present little or no risk of dynamic exchange and lossof nucleic acid ligand because the nucleic acid ligand is covalentlyliked to the particle and/or the steric crowding of ligands slowsdegradation due to nuclease activity. In certain embodiments, themultifunctional nanoparticles of the disclosure as described herein arecapable of differentiating local biochemical environment as a triggerfor therapeutic agent or diagnostic agent release. A major hurdle forcurrent nucleic acid delivery platforms is the endosomal escape, whichis necessary for therapeutic oligonucleotides to be more effective inthe cytosol of cells. Thus, in some embodiments, the degradation ofcertain nanoparticles of the disclosure as described herein results inmodified therapeutic oligonucleotides (e.g., such as hydrophobicallymodified oligonucleotide) capable of escaping the endosomal compartmentsof the cell.

Thus, one aspect of the disclosure provides multifunctionalnanoparticles including one or more of nucleic acid ligands covalentlyattached to a particle including non-polymeric amphiphiles,

-   -   wherein hydrophobic groups of the amphiphiles are arranged        toward the particle interior, and    -   wherein hydrophilic groups of the amphiphiles are at the        particle surface and are crosslinked through a triazole,        thioether, or alkenyl sulfide group with one or more linkers        cleavable by one or more intracellular or extracellular release        agents.

As provided above, the multifunctional nanoparticles of the disclosureinclude particles having non-polymeric amphiphiles. As used herein, theterm “non-polymeric” means a material that is not a polymer (i.e., amolecule composed of repeat units). The amphiphiles of the disclosurehave hydrophobic groups arranged toward the particle interior, andhydrophilic groups are at the particle surface. In certain embodiments,the hydrophobic groups of the amphiphile as otherwise described hereininclude C₆-C₂₂ alkyl, C₆-C₂₂ alkenyl, or C₆-C₂₂ alkynyl group, eachoptionally substituted with halo, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₆alkoxy, or —CO(C₁-C₂₂ alkyl). In other embodiments, the hydrophobicgroups of the amphiphile include unsubstituted C₆-C₂₂ alkyl, C₆-C₂₂alkenyl, or C₆-C₂₂ alkynyl group. In other embodiments, the hydrophobicgroups of the amphiphile include unsubstituted C₆-C₂₂ alkyl; orunsubstituted C₆-C₂₀ alkyl; or unsubstituted C₆-C₁₈ alkyl; orunsubstituted C₆-C₁₅ alkyl; or unsubstituted C₆-C₁₂ alkyl; orunsubstituted C₆-C₁₀ alkyl; or unsubstituted C₁₀-C₂₂ alkyl; orunsubstituted C₁₀-C₂₀ alkyl; or unsubstituted C₁₀-C₁₈ alkyl; orunsubstituted C₁₀-C₁₅ alkyl; or unsubstituted C₁₂-C₂₂ alkyl; orunsubstituted C₁₂-C₂₀ alkyl; or unsubstituted C₁₂-C₁₈ alkyl; orunsubstituted C₁₂-C₁₅ alkyl. In other embodiments, the hydrophobicgroups of the amphiphile include optionally substituted C₁₀ alkyl. Inother embodiments, the hydrophobic groups of the amphiphile includeunsubstituted C₁₀ alkyl. In certain embodiments, the hydrophilic groupsof the amphiphile as otherwise described herein include an ammoniumgroup

The amphiphiles of the disclosure as otherwise described herein arecrosslinked through a triazole, thioether, or alkenyl sulfide group withone or more linkers. In certain embodiments, amphiphiles of thedisclosure as otherwise described herein are crosslinked through atriazole group. In certain embodiments, amphiphiles of the disclosure asotherwise described herein are crosslinked through a thioether group. Incertain embodiments, amphiphiles of the disclosure as otherwisedescribed herein are crosslinked through an alkenyl sulfide group. Incertain embodiments, the triazole, thioether, or alkenyl sulfidecrosslinking group results from a reaction of alkyne or alkene moiety onthe hydrophilic group of the amphiphile (e.g., on the ammonium group)and an azide or thiol moiety on the linker. In one example, the triazolecrosslinker results from a reaction of alkyne moiety on the hydrophilicgroup of the amphiphile (e.g., on the ammonium group) with an azidemoiety on the linker. In one example, the alkenyl sulfide crosslinkerresults from a reaction of alkyne moiety on the hydrophilic group of theamphiphile (e.g., on the ammonium group) with a thiol moiety on thelinker. In one example, the thioether crosslinker results from areaction of alkene moiety on the hydrophilic group of the amphiphile(e.g., on the ammonium group) with a thiol moiety on the linker. Incertain embodiments, the alkyne or alkene moiety on the hydrophilicgroup of the amphiphile of the disclosure as otherwise described hereinis one or two of prop-2-ynyl or prop-2-enyl; one or two of prop-2-ynyl,or one or two of prop-2-enyl. In certain embodiments, the hydrophilicgroup is tri(prop-2-yn-1-yl)amino or triallylamino. In certainembodiments, the crosslinking group results from a reaction ofprop-2-ynyl. In certain embodiments, the crosslinking group results froma reaction of diallylamino.

The hydrophilic groups of the amphiphiles of the disclosure as otherwisedescribed herein are crosslinked with one or more linkers that arecleavable by one or more intracellular or extracellular release agents.For example, in one embodiment, the linker is cleavable by one or moreenzymes, such as, but not limited to, peptidases, proteases, esterases,or elastases. In one embodiment, the linker is cleavable by an esterase.In certain embodiments, the linkers of the disclosure as otherwisedescribed herein are cleavable by one intracellular or extracellularrelease agent. In certain embodiments, the linkers of the disclosure asotherwise described herein are cleavable by two or more intracellular orextracellular release agents (e.g., wherein the linker comprises two ormore different chemical groups each cleavable by a different releaseagent).

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein include a peptide (for example, cleavable with apeptidase or protease), wherein the peptide is at least two amino acidslong. In certain embodiments, the peptide is at least two amino acidslong. In certain embodiments, at least three amino acids long. Incertain embodiments, at least four amino acids long. In certainembodiments, the peptide is between two and twenty amino acids long; orbetween three and twenty amino acids long; or between four and twentyamino acids long. In some embodiments, the peptide linker comprisesGPLGLAGGERDG (SEQ ID NO:10), GFLG (SEQ ID NO:11), GPMGIAGQ (SEQ IDNO:12), Phe-Leu, Val-Ala, Val-Cit, Val-Lys, Val-Arg, or Phe-Lys. In someembodiments, the peptide linker comprises GPLGLAGGERDG (SEQ ID NO:10),GFLG (SEQ ID NO:11), or GPMGIAGQ (SEQ ID NO:12). In some embodiments,the peptide linker is GPLGLAGGERDG (SEQ ID NO:10), GFLG (SEQ ID NO:11),GPMGIAGQ (SEQ ID NO:12), Phe-Leu, Val-Ala, Val-Cit, Val-Lys, Val-Arg, orPhe-Lys. In some embodiments, the peptide linker is GPLGLAGGERDG (SEQ IDNO:10), GFLG (SEQ ID NO:11), or GPMGIAGQ (SEQ ID NO:12). In someembodiments, the peptide linker as otherwise described herein comprises(or further comprises) two Cys groups (for example, at each end of thepeptide linker, such that the sulfur on the peptide linker makes upthioether or alkenyl sulfide group crosslinking the hydrophilic group ofthe amphiphile and the linker).

In some embodiments, the linker of the disclosure as otherwise describedherein comprises GPLGLAGGERDG (SEQ ID NO:10) or GFLG (SEQ ID NO:11). Insome embodiments, the linker of the disclosure as otherwise describedherein is GPLGLAGGERDG (SEQ ID NO:10) or GFLG (SEQ ID NO:11).

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein include one or more of ester groups (for example,cleavable with an esterase). In one embodiment, the linkers of thedisclosure as otherwise described herein include

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein include one or more of hydrazone, semicarbazone,thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, orthioether groups, or a combination thereof, or other acid-labile groupsthat are hydrolyzable in the lysosome.

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein include at least two groups selected from an ester,hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, thioether, disulfide, and a peptide, whereinthe peptide is at least two amino acids long, or at least three aminoacids long, or at least four amino acids long; or the peptide is betweentwo and twenty amino acids long. In certain embodiments, the linker iscleavable by two or more intracellular or extracellular release agentsand include at least two groups selected from an ester, hydrazone,semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester,acetal, ketal, thioether, disulfide, and a peptide as described herein.

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein include a disulfide group.

In certain embodiments, the linkers of the disclosure as otherwisedescribed herein exclude disulfide group or another group cleavableunder reducing conditions.

In certain embodiments, the non-polymeric amphiphiles of the disclosureas otherwise described herein are derived from

wherein X is halogen (e.g., Br). For example, in certain embodiments,the non-polymeric amphiphiles are derived from

wherein X is halogen (e.g., Br).

As provided above, the multifunctional nanoparticles of the disclosureinclude one or more nucleic acid ligands covalently attached to theparticle as otherwise described herein. For example, in someembodiments, the nucleic acid ligands of the disclosure are capable ofselectively binding to a cell surface antigen (aptamer).

In some embodiments, the nucleic acid ligands of the disclosure arecapable of selectively binding to a protein or a carbohydrate. In someembodiments, the nucleic acid ligands of the disclosure are capable ofselectively binding to a protein, wherein the protein is selected fromthe group consisting of tumor-markers, integrins, cell surfacereceptors, transmembrane proteins, ion channels, membrane transportprotein, enzymes, antibodies, and chimeric proteins. In someembodiments, the nucleic acid ligands of the disclosure are capable ofselectively binding to a carbohydrate, wherein the carbohydrate isselected from the group consisting of glycoproteins, sugar residues, andglycocalyx.

In certain embodiments, the nucleic acid ligands of the disclosure asotherwise described herein are capable of selectively binding DNA, RNA,modified DNA, modified RNA, DNAzymes, ribozymes, mRNA, siRNA, microRNA,shRNA, and combinations thereof.

In certain embodiments, the nucleic acid ligands of the disclosure asotherwise described herein are capable of selectively binding to a cellduring a specific developmental stage (e.g., stage havingdevelopmentally specific cell surface antigens) or to a cell in aspecific disease state (e.g., a tumor cell that has tumor-associatedantigens or tumor-specific antigens.)

In certain embodiments, the nucleic acid ligands of the disclosure asotherwise described herein are capable of gene regulation. For example,in some embodiments, the nucleic acid ligands capable of gene regulationcan be siRNA, DNAzyme, ribozyme, microRNA, or any other therapeuticoligonucleotides (including antisense oligonucleotides).

In certain embodiments, the nucleic acid ligands of the disclosure asotherwise described herein can be native or modified, includingphosphorthioated backbones, and 2′ prime protected ribonucleic acids, orcan be an aptamer, either RNA or DNA, modified or unmodified.

The inventors have recognized that, in certain embodiments, themultifunctional nanoparticle of the disclosure can transport the nucleicacid ligand to the cytosol. Without being bound by a particular theory,it is believed that the nucleic acid ligand may be assisted in itsability to reach the cytosol due to its covalent attachment to theamphiphiles (i.e., its relationship to the particle's hydrophobic groupof the amphiphiles).

As provided above, the nucleic acid ligands of the disclosure arecovalently attached to the hydrophilic groups of the amphiphiles. Incertain embodiments, up to two nucleic acid molecules are attached tothe hydrophilic groups of the amphiphiles (e.g., up to two per alkyne).In certain embodiments, one nucleic acid molecule is attached to thehydrophilic groups of the amphiphiles. In certain embodiments, twonucleic acid molecules are attached to the hydrophilic groups of theamphiphiles.

The nucleic acid ligands, for example in certain embodiments, arecovalently attached to the hydrophilic groups of the amphiphiles througha thioether or alkenyl sulfide group. Such thioether or alkenyl sulfidegroups may result from a reaction of alkyne or alkene moiety on thehydrophilic group of the amphiphile (e.g., on the ammonium group) and athiol moiety (e.g., Cys) on the nucleic acid ligand. In certainembodiments, the alkyne or alkene moiety on the hydrophilic groups ofthe amphiphiles is prop-2-ynyl or prop-2-enyl, or prop-2-ynyl, orprop-2-enyl; or the alkyne or alkene moiety on the hydrophilic group ofthe amphiphile is prop-2-yn-1-ylamino or allylamino.

The multifunctional nanoparticles as described herein can be provided ina variety of different particle sizes, depending, e.g., on theamphiphiles and crosslinkers used for making them. For example, incertain embodiments, the multifunctional nanoparticle as describedherein has a particle size within the range of about 0.1 nm to about 1μm in diameter, e.g., 1 nm to 500 nm, or 1 nm to 100 nm, or 1 nm to 50nm, or 1 nm to 30 nm, or 1 nm to 20 nm, or 1 nm to 10 nm, or 10 nm to 1μm, or 10 nm to 500 nm, or 10 nm to 100 nm, or 10 nm to 50 nm, or 10 nmto 30 nm, or 10 nm to 20 nm, or 20 nm to 500 nm, or 20 nm to 100 nm, or20 nm to 50 nm, or 20 nm to 40 nm, or 50 nm to 500 nm, or 50 nm to 100nm in diameter. In certain embodiments, the multifunctional nanoparticleas described herein has a particle size within the range of about 10 nmto about 100 nm in diameter. The person of ordinary skill in the artcan, in view of the materials and methods described herein, provide adesired particle size to a multifunctional nanoparticle.

In certain embodiments, the multifunctional nanoparticles as describedherein have a discrete particle size and are monodisperse (i.e.,uniform).

Another aspect of the disclosure provides conjugates comprising themultifunctional nanoparticle of the disclosure as otherwise describedherein and at least one therapeutic agent or diagnostic agent, whereinthe multifunctional nanoparticle encapsulates the therapeutic agent.

In certain embodiments, the conjugate as otherwise described hereinincludes a therapeutic agent. The therapeutic agent may be a hydrophobicsmall molecule drug, such as, but not limited to, an anti-cancer agent,an antibiotic, an antiviral, an antiparasitic agent, an anticoagulant,an analgesic agent, an anesthetic agent, an ion channel potentiator, anion channel inhibitor, an anti-inflammatory, a metallodrug, and anycombination thereof. For example, in certain embodiments, thetherapeutic agent is selected from camptothecin, doxorubicin,daunorubicin, vincristine, paclitaxel, neocarzinostatin, calicheamicin,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,lurtotecan, annamycin, docetaxel, tamoxifen, epirubicin, methotrexate,vinblastin, vincristin, topotecan, prednisone, prednisolone, andabt-737.

In certain embodiments, the conjugate as otherwise described hereinincludes a diagnostic agent. The diagnostic agent may be, for example, afluorophore, a radiolabeled nucleotide, a radioisotope, biotin,tocopherol, cholesterol, a steroid, or a electron dense tag and a metalchelator.

In certain embodiments, the conjugate as otherwise described hereinincludes a therapeutic or a diagnostic agent in the amount ranging from0.25% to 10%, relative to the concentration of the non-polymericamphiphile. In certain embodiments, the therapeutic or the diagnosticagent is present in the amount from 1% to 5%, or from 1% to 4%, or from1% to 3%, or from 1% to 2%, or from 2% to 5%, or from 2% to 4%, or from2% to 3%, or about 2.5%, relative to the concentration of thenon-polymeric amphiphile.

As a person of skill in the art will recognize, the multifunctionalnanoparticles of the disclosure or the conjugates of the disclosure maybe provided in a pharmaceutical composition. For example, themultifunctional nanoparticles of the disclosure or the conjugates of thedisclosure may be provided together with at least one pharmaceuticallyacceptable carrier, diluent, and/or excipient. The exact nature of thecarrier, excipient or diluent will depend upon the desired use for thenanoparticles or conjugates, and may range from being suitable oracceptable for veterinary uses to being suitable or acceptable for humanuse. The composition may optionally include one or more additionaltherapeutic agents and/or diagnostic agents as described herein.

Another aspect of the disclosure provides methods of treating a diseaseor disorder, including administering to a subject in need thereof aneffective amount of the conjugate of the disclosure, wherein the linkeris cleavable by one or more intracellular or extracellular release agentpresent in the subject, thus releasing the therapeutic agent ordiagnostic agent.

For example, in some embodiments, the disease or disorder is cancer,infection (e.g., bacterial, viral, or parasitic), pain, asthma,inflammation, neurological disease or disorder (e.g., Alzheimer'sdisease, Parkinson's disease, etc.). In certain embodiments, the diseaseor disorder is asthma, inflammation (e.g., asthma-induced inflammationor chronic obstructive pulmonary disease (COPD)-induced inflammation),infection (e.g., lower respiratory infections), or cancer.

In certain embodiments of the methods of the disclosure, the conjugatecomprises a therapeutic agent as described herein.

The linkers of the disclosure may be selectively cleaved. For example,in one embodiment, no more than about 20%, no more than about 15%, nomore than about 10%, no more than about 5%, no more than about 3%, or nomore than about 1% of the linker is cleaved in an extracellularenvironment. In another embodiment, no less than about 20%, no less thanabout 15%, no less than about 10%, no less than about 5%, no less thanabout 3%, or no less than about 1% of the linker is cleaved in anextracellular environment.

In certain embodiments of the methods of the disclosure, the releasemechanism is an enzyme expressed by tumor cells.

In certain embodiments of the methods of the disclosure, the releaseagent is a lysosome agent, endosome agent, and/or caveolae agent.

Definitions

The following terms and expressions used herein have the indicatedmeanings.

Substituents are intended to be read “left to right” (i.e., theattachment is via the last portion of the name) unless a dash indicatesotherwise. For example, C₁-C₆ alkoxycarbonyl and —C(O)C₁-C₆alkylindicate the same functionality; similarly arylalkyl and -alkylarylindicate the same functionality.

The term “alkenyl” as used herein, means a straight or branched chainhydrocarbon containing from 2 to 10 carbons, unless otherwise specified,and containing at least one carbon-carbon double bond. Representativeexamples of alkenyl include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and3,7-dimethylocta-2,6-dienyl.

The term “alkoxy” as used herein, means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms unless otherwisespecified. Representative examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl. When an “alkyl” group is a linking group between two othermoieties, then it may also be a straight or branched chain; examplesinclude, but are not limited to —CH₂—, —CH₂CH₂—, —CH₂CH₂CHC(CH₃)—, and—CH₂CH(CH₂CH₃)CH₂—.

The term “alkynyl” as used herein, means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “halo” or “halogen” as used herein, means —Cl, —Br, —I or —F.

Certain aspects of the disclosure are now explained further via thefollowing non-limiting examples.

Examples Example 1: Preparation and Evaluation of Ester-Crosslinked NANs

General Method:

For the purposes of synthesis and spectroscopic analyses, methylenechloride, methanol, hexanes, tetrahydrofuran, dimethylformamide,acetonitrile, and ethyl acetate were of HPLC grade. All other reagentsand solvents were of ACS-certified grade or higher, and were used asreceived from commercial suppliers. ¹H and ¹³C NMR spectra were recordedon a Bruker DRX-300 spectrometer. Mass spectrometry analysis wasrecorded on a Sciex QSTAR Elite mass spectrometer.

Synthesis of Surfactant 1 (N,N,N-tri(prop-2-yn-1-yl)dodecan-1-aminiumbromide)

To a solution of dodecylamine (50 mg, 0.27 mmol) in 10 mL of methanol,anhydrous potassium bicarbonate (90 mg, 0.64 mmol) was added followed bydropwise addition of propargyl bromide (67 mg, 0.57 mmol) for a periodof one minute. The mixture was stirred at room temperature for 12 hoursafter which propargyl bromide (34 mg, 0.29 mmol) was added and thesolution stirred further at 40° C. for 6 hours. The mixture was cooledto room temperature and filtered. The solvents were removed from thefiltrate and the concentrated sample purified by column chromatographyover silica gel using methanol/methylene chloride (1:15) as eluent toyield the product as white powder (70 mg, 68%). ¹H NMR (300 MHz, CDCl₃,δ): 4.80 (d, J=2.13 Hz, 6H), 3.7 (t, J=8.7 Hz, 2H), 3.04 (s, 3H), 2.01(s, 2H), 1.40-1.20 (m, 18H), 0.9 (t, J=6.60 Hz, 3H). ¹³C NMR (300 MHz,CDCl₃, δ): 82.3, 83.3, 83.3, 69.8, 69.8, 69.8, 60.4, 49.9, 49.9, 49.9,31.9, 29.7, 29.6, 29.4, 29.4, 29.3, 28.8, 26.2, 22.7, 22.4, 14.1.ESI-HRMS (m/z): [M-Br]+ calculated for C₂₁H₃₄N, 300.2691; found,300.2664.

Synthesis of Ester-Crosslinked NAN

In the typical procedure illustrated in FIG. 1, micelle solution ofsurfactant 1 (3.8 mg, 0.01 mmol) was prepared in Millipore water (1 mL).To the solution, ethane-1,2-diyl bis(2-azidoacetate) (diazidecrosslinker 2; 2.8 mg, 0.012 mmol; prepared as described in Chen et al.Synth. Commun. 1998, 28, 3097), THPTA-Cu complex (0.00025 mmol), andsodium ascorbate (5.0 μL of a 99 mg/mL solution in water, 0.0025 mmol)were added. The reaction mixture was stirred slowly at room temperature.After 4 hours, the sample was purified by Sephadex G-25 Nap-5 column andthe fractions containing crosslinked micelles (CM) were analyzed byUV-Vis, dynamic light scattering (DLS, and zeta potential. In adisposable cuvette, a solution containing the crosslinked micelles (10μM) was prepared. To this solution, DNA anchor (30 μM) and2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (2.5 μM) were addedfor a total volume of 500 μL in Millipore water. The mixture was placedin a Rhyonet reactor for 30 minutes. The thiol-yne photocrosslinking wasmonitored by dynamic light scattering (DLS) and agarose gelelectrophoresis.

Discussion:

The NAN's core is synthesized using a two-step approach combiningself-assembly and a surface cross-linking step. The major difference inthe NAN's core design is in the cross-linking step which can be used totrigger an enzymatic disassembly step, important for biochemicallycontrolled drug release. First, an alkyne-terminated surfactant 1 wasself-assembled in water. After assembly the particles are covalentlycross-linked to hold the micelle-like structure intact. The cross-linker2 is functionalized with azido groups on either end to facilitatecross-linking to the alkyne head groups presented by the surfactant.This cross-linker is incorporated into the particles design in order tostabilize the nanocapsule's core and recruit enzymes for initiating itsdegradation. Esterases are enzymes that can rapidly recognize and cleaveester bonds and have been shown to be effective targets for catalyzingdrug release from nanomaterials. In addition to stabilizing the particlewith ester linkages the NANs gain the potential to release hydrophobicsmall molecules from their core in response to enzymes (FIG. 1).

Esterases were also specifically chosen as the biochemical trigger fordegrading the nanocapsules as these enzymes are known to be concentratedinside cellular endosomes. As many drugs and therapeutic antisenseoligonucleotides need to reach the cytosol to exert their therapeuticeffect, it was of interest to release the contents of the NAN onceinside the cell in order to increase the likelihood its contents wouldmake it to the cytosol.

Through careful control over the stoichiometry of the diazidocross-linker relative to the total number of alkynes presented at theparticle surface (1:1.2 respectively), enough alkynes could be leftunreacted to allow for attachment of a thiolated nucleic acid in thesecond assembly step. Using this approach thiolated DNA was attached tothe surface of the nanocapsule using UV irradiation (365 nm) and awater-soluble photoinitiator traditionally used in polymerizationreactions.

The resulting NAN is monitored during each step of assembly through aseries of characterization techniques including dynamic light scattering(DLS), transmission electron microscopy (TEM) and zeta potentialmeasurements (FIG. 2). The cross-linked micelle (CM) prior to DNAconjugation exhibited a uniform size distribution (20±3 nm) and positivecharge (+50±4 mV). Such a positive surface charge was expected due tothe presence of the tertiary amines presented by the alkynefunctionalized headgroup. Post DNA attachment, the particles exhibited adramatic shift in surface charge to −40 mV (FIG. 3A). The particle sizealso increased from 20 to 34 nm as would be expected for the attachmentof a 22mer DNA strand (FIG. 2B). Additionally, post DNAfunctionalization, the once highly positively charged nanocapsule couldnow migrate through a 1% agarose gel under an electric field, indicativeof the presence of the negatively charged DNA strands at the particlessurface (FIG. 3B).

Evaluation of Release Ability:

To determine the ability of NANs to release a small molecule from itsinterior, NANs were synthesized in the presence of rhodamine B dye. Theeffective loading of the particles was optimized at 2.5% loading byconcentration although loading as high as 10% was possible (results notshown). The resulting particles were then subjected to treatment withporcine liver esterase and monitored using fluorescence spectroscopy. Inshort, A solution of NANs (3 μM) was prepared in Tris HCl buffer in atotal volume of 400 μl. The solution was placed into an external Peltierunit and allowed to come to 37° C. Esterase (Porcine liver esterase,Sigma Aldrich, 5 units) was added and a reading was taken immediately.All samples were excited at 545 nm and scanned between 570 and 700 nmusing a Jobin Yvon Fluorometer while heated at 37° C. This process wasrepeated at 5, 15, 30, 45, 60, 90, 120, 180, 210, 240, 270, and 300minutes. The NANs containing ester linkages were successfully cleaved asindicated by an increase in the samples fluorescence over time (FIG.4A). As a control, a nanocapsule cross-linked with non-ester linkage wasprepared by reacting surfactant 1 with 1,4-diazidobutane-2,3-diol (3).This nanocapsule cross-linked with non-ester linkage was treated withesterase and release of the dye was not observed (FIG. 4B). The lack ofdye release indicates that the stability of the particle is gated by theenzymatic environment of the nanocapsule. The NANs were also evaluatedfor their cell toxicity effects and shown to be nontoxic up tomicromolar concentrations (FIG. 4E).

Next, cells were incubated with the NANs to see if the SNA-likeconstruct to determine cellular uptake without transfection agents, afeature exhibited by SNA configurations on nanoparticle surfaces.Confocal studies showed that the particles were indeed readily taken upby cells in a fashion similar to that seen with traditional SNA-likestructures (see FIG. 5C-F), and were shown to colocalize within regionsof the cell that indicate endocytosis as the main mechanism of uptake.

Example 2: Preparation of Drug-Loaded Ester-Crosslinked NANs

Ligation of DNAzyme/Mutated DNAzyme to Nucleic Acid Nanoparticle (NAN):

10 μM GATA-3 DNAzyme/mutated DNAzyme and 20 μM GATA-3 DNAzymebridge/mutated DNAzyme bridge were added to 200 μL of 4 μM solution ofNANs of Example 1 functionalized with anchor. Water was added to thesample to reach a total volume of 300 μL. The solution was heated at 70°C. for 10 minutes and cooled to room temperature. 5 mM ATP, 15 μL of 1U/μL T4 DNA Ligase (Invitrogen), 1× ligase buffer were mixed. Water wasadded to this second solution to reach a total volume of 300 μL. Placedon 25° C. heat block for 2 hours, ligase was heat inactivated at 65° C.for 10 minutes.

TABLE 1 DNAzyme-NAN characterization and activity assaysDNA and RNA sequences  Anchor  5′-SH-TTT TTT TTT TCA CGT CCA GCA G-3′(AKH-anchor) (SEQ ID NO: 1) GATA-3   ** DNAzyme5′-GTG GAT GGA CCC TAG CTA CAA CGA CTC  TTG GAG-3 (SEQ ID NO: 2)  GATA-35′-GCC TCC ATC CAC CTG CTG GAC CTC-3′  DNAzyme (SEQ ID NO: 3) bridge   ***   *  Mutated 5′-GCG GCT GGA CCC TAG CTA CAA CGA CTC DNAzyme  *  *TCG TAG-3′ (SEQ  ID NO: 4)  Mutated5′- GCC TCC AGC CCC CTG CTG GAC CTC-3′  DNAzyme (SEQ ID NO: 5) bridge GATA-3 mRNA  5′-Cy3-CUC CAA GAC GUC CAU CCA C-3′  truncate 1(SEQ ID NO: 6) GATA-3 mRNA 5′ FAM- CUC CAA GAC GUC CAU CCA C - truncate 2  BHQ-13′ (SEQ ID NO: 7) * indicates mutations, ** indicatesmonophosphorylated site. Note: mutations in DNAzyme affect the flankingregion of the DNAzyme to test for target specificity. No changes weremade to the catalytic loop region associated with cleavage.

Cleavage Assay.

To test the activity of the GATA-3 DNAzyme, both free DNAzyme and 5 μMof ligated NANs were incubated with 0.5 μM GATA-3 mRNA truncate, both inthe presence and absence of salts (10 mM MgCl₂ and 100 mM NaCl). Thedifferent reactions were then run on an 8% denaturing polyacrylamide gelat 350 V for 30 minutes. The gel was scanned using both a 473 nm laserand a 532 nm laser.

Cellular Uptake and Confocal Imaging of DNAyzme-NANs.

HeLa cells were grown in 10% FBS in DMEM with 1%Penicillin/Streptomycin. Confluent cells were treated with 1 μMfunctionalized NANs for 3.5 hours, then washed with 1×PBS. Media wasreplaced and cells were imaged using a Leica SP8 confocal microscope.

Ligated DNAzyme and Fluorescent mRNA Probe Experiments.

The interaction between ligated DNAzyme-NANs and a dually labeled blackhole quencher (BHQ) and dye (FAM) labeled mRNA truncate was investigatedusing a Horiba Yvon flurolog-3 fluorometer.

A solution of MgCl₂ (10 mM), NaCl (100 mM), ligated DNAzyme NANs (100nM) and di H₂O was used as a control to determine the auto fluorescencefrom the NAN and salt solution. Prior to mixing, the ligated DNAzyme NANwas heated to 70° C. for ten minutes and then cooled to roomtemperature. The control sample was then held at 37° C. in a HoribaJobin Yvon fluorometer using an external Cary single cell peltieraccessory. The control sample was excited at 470 nm and scanned from 485to 700 nm.

A second sample was prepared this time containing the BHQ FAM mRNAtruncate. A solution of MgCl₂ (10 mM), NaCl (100 mM), ligated DNAzymeNAN (100 nM) and H2O was prepared as above, with the ligated DNAzyme NANbeing heated to 70° C. for ten minutes prior to mixing. The sample wascooled to and held at 37° C. in the Horiba Jobin Yvon fluorometer usingan external Cary single cell peltier accessory. The dually labeledBHQ-mRNA-FAM (10 nm, BioSearch Technologies) was added to the sample anda measurement was taken immediately. The sample was excited at 470 nmand scanned between 485 and 700 nm. Further measurements were taken at2, 5, 10, 15, 20, 30, 45, 60 minutes.

Discussion:

The NANs of Example 1 were loaded with camptothecin, an apoptosisinducing drug and incubated with HeLa cells for 4 h at 37° C. Cellproliferation studies showed that cells treated with camptothecin-loadedNANs (50 μM drug) effectively limited the growth of cells by 50%relative to untreated cells. Non-ester cross-linked NANs loaded with 50μM drug had minimal effect on cell growth (FIG. 4D).

A fully degradable aspect of the NAN construct was evaluated through theattachment of a DNAzyme that requires its folded structure to functionin the cleavage of mRNA. The DNAzyme hgd40 was specifically chosen as atest oligonucleotide sequence as it has recently been shown to rapidlycleave mRNA encoding an important transcription factor (GATA-3) involvedin inflammation pathways. In order to design this construct the DNAzymewas first enzymatically assembled onto the NANs surface using a recentlydeveloped enzyme ligation approach compatible with SNA like structuresas described by Rouge et al. (ACS Nano 2014, 8, 8837).

The DNAzyme-functionalized NANs were first incubated with an mRNAtruncate of GATA-3 and then evaluated for evidence of mRNA cleavage invitro. The DNAzyme-functionalized NANs were shown to be effective atcleaving a truncated GATA-3 mRNA sequence at 37° C. after 4 h asindicated by polyacrylamide gel electrophoresis (FIG. 5A). To probe therelative amount of cleavage of the mRNA truncate in the presence of theDNAzyme-functionalized NAN, a modified mRNA truncate containing a dye(FAM) and a dye quencher (BHQ-1) was monitored using fluorescencespectroscopy. In the presence of 100 nM DNAzyme-NANs, 10 nM mRNAtruncate is fully cleaved within the first hour of incubation asindicated by the increase in fluorescence observed (FIG. 5B). Theseresults indicate that the particle surface can be easily modified usingenzymes with functional, therapeutic nucleic acids.

Example 3: Preparation and Evaluation of Peptide-Crosslinked NANs

Peptide Surface Crosslinked Micelles (pep-SCMs):

A typical procedure is illustrated in FIG. 6. For example, 1.9 mg ofsurfactant 1 was dissolved in Millipore water (500 μL). 31.25 μL of a 4mM 5-carboxytetramethylrhodamine (5-TAMRA) stock solution in DMSO wasadded to the micelle solution and allowed to stir for 30 minutes at roomtemperature. 17 μL of either CGFLGC (cathepsin B substrate) (SEQ IDNO:8) or CGPLGLAGGERDGC (MMP9 substrate) (SEQ ID NO:9) was added to 232μL of the micelle solution. 1 μL of2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (DHEMPP) was alsoadded, resulting in the following final concentrations: surfactant (9.28mM), 5-TAMRA (250 μL), peptide crosslinker (5 mM) and DHEMPP (20 μM).The final solution was placed in a Rhyonet reactor for 30 minutes. Theproduct was characterized by DLS and zeta potential on a ZetasizerNano-ZS90, by TEM, and by 1% agarose gel electrophoresis.

Peptide Nucleic Acid Nanocapsules (pep-NANs):

5 μL of pep-SCMs was diluted to a total volume of 500 μL to give aconcentration of 92.8 μM. Included in this dilution was 38.5 μL of athiolated DNA (100 μM) and 1 μL of2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (20 μM). The finalsolution was placed in a Rhyonet reactor for 30 minutes. The product waspurified by Sephadex G-25 NAP-10 column. The product was characterizedby DLS and zeta potential on a Zetasizer Nano-ZS90, TEM, and by 1%agarose gel electrophoresis.

Example 4: Preparation and Evaluation of Gold Nanoparticle EncapsulatedPeptide-Crosslinked NANs

Gold Nanoparticle Encapsulated Peptide Surface Crosslinked Micelles(pep-Au-SCMs):

A typical procedure is illustrated in FIG. 7. For example, 10 μL oftetradecylamine functionalized gold nanoparticles in cyclohexanes wereair dried. 1.5 mg of surfactant was added to 500 μL of water and 20 μLof hexanes giving a final concentration of 7.62 mM by surfactant. Thissolution was allowed to stir overnight. The solution was diluted to 1 mLgiving a new concentration of 3.81 mM by surfactant. 12 μL of eitherCGFLGC (cathepsin B substrate) (SEQ ID NO:8) or CGPLGLAGGERDGC (MMP9substrate) (SEQ ID NO:9) was added to 487 μL of the AuNP micellesolution. 1 μL of DHEMPP was also added, giving these finalconcentrations: surfactant (3.71 mM), peptide crosslinker (2 mM) andDHEMPP (20 μM). The final solution was placed in a Rhyonet reactor for30 minutes. The product was centrifuged for 15 minutes at 8,000 rpm andthe supernatant was removed. This was repeated 3 times and thepep-Au-SCMs were reconstituted in 250 μL of Millipore water. The productwas characterized using DLS, zeta potential measurements, and TEM.

Gold Nanoparticle Encapsulated Peptide Nucleic Acid Nanocapsules(pep-Au-NANs):

6 μL of pep-Au-SCMs was diluted to a total volume of 500 μL to give aconcentration of 100 uM. Included in this dilution was 38.5 μL ofthiolated DNA (100 μM) and 1 μL of2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (20 μM). The finalsolution was placed in a Rhyonet reactor for 30 minutes. The product wascentrifuged for 15 minutes at 8,000 rpm and the supernatant was removed.This was repeated 3 times and the pep-Au-NANs were reconstituted in 250μL of Millipore water. The product was characterized using dynamic lightscattering, zeta potential, and transmission electron microscopy.

Gold Nanoparticle Encapsulated Nucleic Acid Nanocapsules(Diol/Ester-Au-NANs): A modified protocol from above was used tosynthesize gold encapsulated diol and ester NANs. Gold encapsulated diolNANs was prepared by reacting surfactant 1 with1,4-diazidobutane-2,3-diol (3). Gold encapsulated ester NANs wasprepared by reacting surfactant 1 with diazide crosslinker 2.

Discussion:

Many sensing applications attempt to determine the presence of enzymesexpressed, which can vary dramatically during the course of a cell'slife, and differ depending on different disease states. With this inmind, well-studied cathepsin B and MMP9 were tested. In addition totheir implication in several cancer types, importantly, MMP9 is locatedin the extracellular matrix (ECM) of cells, and functions optimally atphysiological pH (pH 7), whereas cathepsin B is an endosomal proteasethat functions optimally at pH 5. As a control the localization of theseenzyme-responsive peptide cross-linked-nucleic acid nanocapsules(pep-NANs) was compared to the activity of ester-crosslinked andnon-ester, diol crosslinked NANs of Example 1 using electron microscopy.

By introducing a peptide substrate whose sequence was modified at boththe N and C terminus with cysteine residues to allow for chemicalattachment points (thiols) for reacting with the alkynes presented atthe surface of the NAN's micellular core (FIG. 8). Characterization ofthe successful assembly of the pep-NANs was achieved using a combinationof dynamic light scattering (DLS), transmission electron microscopy(TEM), zeta potential measurements, and gel electrophoresis (FIG. 9).Collectively these results indicated nanocapsule populationsapproximately 35 and 24 nm in size for cathepsin B peptide substratelinked NANs (CathB-NANs) and matrix metalloproteinase 9 peptidesubstrate linked NANs (MMP9-NANs), respectively (FIG. 9B, Table 2).

TABLE 2 Average pep-NAN DLS and zeta potential data. CathB- CathB-Au-MMP9- MMP9- MMP9-Au- Measurement SCM CathB-NAN NAN SCM NAN NAN DLS 30.2± 2.7  34.8 ± 5.2  30.2 ± 4.8 27.3 ± 2.4  23.6 ± 2.0  33.1 ± 1.8 ZetaPotential 47.0 ± 2.5 −30.8 ± 6.6 −41.9 ± 2.8 41.6 ± 2.6 −37.9 ± 6.7−45.4 ± 2.1 SCM = surface crosslinked micelle, NAN = nucleic acidnanocapsule.

A fluorescence assay was used to monitor the rate and extent of dyerelease (FIG. 10) in order to test individual enzyme specificity for thepeptide substrates within each pep-NAN. MMP9-NANs and CathB-NANsreleased their dye when treated with their intended enzyme targets, MMP9and cathepsin B, respectively (FIG. 10 A,B). Cathepsin B activity onMMP9-NANs was also evaluated, in which no fluorescence change wasobserved (FIG. 10C). This indicates that proteases from differentfamilies of enzymes are less likely to recognize the substrates of otherenzymes within the pep-NAN formulations. The results of theseexperiments also indicated that the pH of the solution can control therelease or lack of release of dyes from a pep-NAN formulation despitethe presence of the appropriate enzyme trigger in solution. To test therelative rate of cleavage of the MMP9-NANs in the presence of closelyrelated MMP enzymes, the MMP9-NANs was treated with various MMPproteins, including MMP1 and MMP2 (a collagenase and a gelatinase,respectively), both of which function optimally at pH 7. The results ofthese experiments indicated that the MMP9-NAN is specific to the MMP9enzyme (FIG. 10D). Additional MMP enzymes were also tested, showing asimilar inability to cleave the MMP9-NANs (results not shown).

Dye-loaded pep-NANs were also incubated with HeLa cells and observedunder confocal microscopy (results not shown). Both the CathB and MMP9NANs entered cells readily when incubated in serum free conditions (FIG.11A) indicating that, like other spherical nucleic acid structures, theydo not require addition of external transfection agents. The pep-NANswere also examined in cells over a range of concentrations in order toassess their relative toxicity in cell culture. The resulting cellsurvival indicated little to no toxicity for both the CathB-NANs and theMMP9-NANs indicating their potential use in both in vitro and cellculture settings.

To be able to engage individual populations of enzymes in specificcellular locations for regulating cargo release, it became particularlyimportant to develop a more precise way of tracking the pep-NANsintracellularly in order to be able to anticipate the biochemicalenvironment that they would experience. To visualize the nanocapsulesintracellularly, a protocol for encapsulating gold nanoparticles (AuNPs)into the interior of the pep-NANs was developed. Starting with a 10 nmtetradecylamine-modified AuNP, the addition of surfactant in water wasused to build the NAN's exterior shell around the alkane-functionalizedAuNP (FIG. 11B). The gold nanoparticle-embedded NANs, referred to hereas pep-Au-NANs, were incubated with HeLa cells to reveal theintracellular location post uptake and to determine if the stability ofthe pep-NANs could be visualized (i.e., shell intact or degraded) usinguranyl acetate staining of charged organic species. Using this approach,both pep-NANs and chemically crosslinked NANs could be successfullyvisualized (FIG. 11C,D). By controlling the stoichiometric ratio ofsurfactant, AuNP and cross-linker, roughly 1 AuNP per NAN could beachieved per particle, regardless of cross-linker utilized, as observedby TEM (data not shown).

After characterizing the pep-Au-NANs, they were incubated with HeLacells and subsequently sectioned, stained, and imaged for evidence ofpep-Au-NAN uptake into cells (FIG. 12A-D). As shown in FIG. 12, diverseformulations of the Au-NANs could be found within endosomes with varyingdegrees of stability, within the cross-sectioned HeLa cells. In FIG.12A,B the negative and positive controls for NAN disassembly are shown(negative control is a diazido-diol cross-linker, compound 3, and thepositive control is the ester cross-linker, compound 2). As anticipated,the diazido-diol cross-linked NANs could not release their cargo in theendosomes, and the ester-cross-linked NANs could, giving us an image ofwhat an intact and degraded NAN shell looks like intracellularly,respectively. This interpretation is based on the fact that an intactgray halo, similar to those seen in FIG. 11C,D, is clearly seensurrounding each of the AuNPs in the endosomal compartments in FIG. 12A.In addition, the lack of organic stained halo seen in FIG. 12B alongwith the aggregated nature of the AuNPs suggests the loss of surfacefunctionalization.

The results of these studies indicate that the NAN's outer structurecould indeed dictate the release of the cargo from the nanocapsule. Thiswas seen in the context of both the chemically cross-linked as well aspeptide-cross-linked NANs. Importantly, the MMP9-NANs did not opendespite being endocytosed, which correlates well with the fact that MMP9enzyme is not expressed intracellularly, nor secreted in significantlevels by HeLa cells. Cathepsin B, although found in HeLa cells, did notopen the CathB-NANs, potentially due to the incubation time within theHeLa cells might not have been long enough to result in the breakdown ofthe pep-NANs.

The later reasoning is thought to be the case based on the degradationrate observed in vitro in the fluorescence assay results (results notshown). The results show it takes ˜20× as long to see comparablefluorescence changes in solution when cathepsin B is added to a solutionof CathB-NANs as compared to MMP9-NAN degradation by MMP9. Additionally,it is important to consider that enzyme concentrations rise and falldepending on the current level of mRNA expression, and can bedysregulated (often overexpressed) when cells become diseased.

In order to test the correlation between enzyme expression level withina live cell and the ability of the pep-NANs to be degraded, anadditional assay was run in which HeLa cells were treated with phorbol12-myristate 13-acetate (PMA), a known inducer of MMP9 expression inHeLa cells. Cells were incubated in serum-free media and treated withMMP9-NANs carrying camptothecin, a known apoptotic cancer drug. Thetoxicity of the drug loaded MMP9-NANs was evaluated using an MTS assaypost 21 h PMA treatment, allowing enough time for MMP9 expression. Thesestudies resulted in a dose dependent increase in cellular toxicity,interpreted as a response to the PMA inducing MMP9 expression andsecretion by the HeLa cells, ultimately catalyzing the release of thedrug in response to the pep-NAN's enzyme target (FIG. 12E,F). Treatmentof PMA and drug loaded MMP9-NANs without allowing time for MMP9secretion resulted in no cell toxicity.

In conclusion, the peptide-NANs were found to have important advantagesin controlling the release of cargo in cell specific locations. Thisadded level of control and specificity over degradation and release ofinternalized cargo, coupled with the rapid and modular nature of theassembly approach, offers significant advantages of the multifunctionalnanoparticles of the disclosure. In one embodiment, for example, thehybrid peptide-based multifunctional nanoparticles of the disclosure aresuitable for various therapeutic and diagnostic applications.

Example 5: Preparation and Evaluation of DNAzyme Ester-Crosslinked NANs(DNAzyme-NAN)

GATA-3 targeting DNAzyme (hgd40) (Sel et al. J. Allergy Clin. Immunol.2008, 121, 910-916; Krug et al. N. Engl. J. Med. 2015, 372, 1987-1995.)was chosen as the proof of concept sequence for evaluating the generegulation capabilities of the DNA surfactants in cell culture for itswell-studied activity in vitro and in vivo using traditional deliverymethodologies. The GATA-3 gene is of particular biological importance asit regulates downstream inflammation responses in immune cells. It hasbeen a target gene of interest for diseases such as asthma and chronicinflammatory diseases.

Nucleic Acid Nanocapsule (NAN) Synthesis:

1.9 mg of surfactant 1 (0.005 mmol) was dissolved in 483.2 μL Milliporewater. Solution was stirred at room temperature for 30 minutes. 10 μL ofa 25 mg/mL sodium ascorbate solution (0.00125 mmol), 5 μL of a 25 mMTHPTA-Cu complex (0.000125 mmol), and 1.8 μL esterified diazidocross-linker 2 (0.006 mmol) were added to a total volume of 500 μL.Mixture was stirred at room temperature for 3.5 hours. The product waspurified by a Sephadex G-25 NAP-10 column (GE Healthcare), and thefractions containing surface crosslinked micelles (SCMs) werecharacterized through dynamic light scattering (DLS) and zeta potential.A solution containing 100 μM SCMs, 150 μM thiolated DNA, and 20 μM of2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (DHEMPP) in a totalvolume of 500 μL was placed in a Rhyonet reactor for 30 minutes. Theresulting product was purified by a Sephadex G-25 NAP-10 column, and thefractions were characterized by DLS and zeta potential measurements on aMalvern Zetasizer Nano Z590.

Ligation of DNAzyme to Nucleic Acid Nanocapsule (NAN):

20 μM GATA-3 DNAzyme (SEQ ID NO:2) and 40 μM DNAzyme bridge (SEQ IDNO:3) were added to 10 μM NANs functionalized with DNA anchor (SEQ IDNO:1). Water was added to a total volume of 300 μL. The solution washeated at 70 μC for 10 minutes and cooled to room temperature. 5 mM ATP,15 μL of 1 U/μL T4 DNA Ligase (Invitrogen), and 1× ligase buffer weremixed. Water was added to a total volume of 300 μL. Placed on a 25° C.heat block for 2 hours. Ligase was heat inactivated at 65° C. for 10minutes. Product was washed through a Sephadex G-25 NAP-10 column.Fractions were then analyzed by DLS to purify unligated DNAzyme fromDNz-NANs.

DNAzyme Stability Assay:

To determine the stability of the DNAzyme ligands on the NANs, 1.5 μMfree DNAzyme and 1 μM DNz-NANs were incubated in either phosphatebuffered saline (PBS, VWR International) or 10% fetal bovine serum (FBS,ThermoFisher Scientific) for 1 hour at 37° C. After incubation, productswere amplified through 35 cycles of PCR and run on an 8% denaturingpolyacrylamide gel.

Quantification of DNAzymes/NAN:

Equivalent volumes of 8 M urea and TYE563-DNAzyme functionalized NANswere heated at 70° C. for 10 minutes and purified by a Sephadex G-25NAP-10 column. Fluorometric measurements were taken of fraction 3 with aHoriba Jobin Yvon Fluorolog 3 series fluorometer. Excitation: 549 nm.Scan: 563-625 nm. TYE563-DNAzyme values were compared to a standardcurve to obtain the final DNAzyme concentration. Typical startingconcentrations of DNAzyme-NANs were 125 nM or 250 nM.

Kinetics Cleavage Assay:

To determine the kinetics of the cleavage of GATA-3 by both the free andNAN-bound DNAzyme, a fluorescence-based assay was developed. A mRNAtruncate corresponding to the cleavable sequence in GATA-3 mRNA waspurchased (BioSearch Technologies). The truncate, herein referred to asBHQ-mRNA, was functionalized with a quencher (BHQ1) at the 3′ end and afluorescein derivative (FAM) at the 5′ end. BHQ-mRNA was shown to bestable at relevant salt concentrations [MgCl2 (10 mM), NaCl (100 nM)]and at the relevant temperature (37° C.) and was therefore deemedsuitable for the kinetics study. All fluorescence measurements weretaken using a Horiba Jobin Yvon fluorometer and, unless otherwisestated, all samples were excited at 470 nm and measurements taken from485 nm to 4700 nm. Samples were heated to their stated temperature usinga Cary single cell peltier accessory. A solution of MgCl2 (10 mM), NaCl(100 mM), DNAzyme (10 nM) and H₂O was prepared in a total volume 400 μL.The DNAzyme solution was heated to 70° C. for ten minutes prior tomixing. The sample was cooled to and held at 37° C. in the fluorometerand an initial background reading was taken. BHQ mRNA (10 nM, BioSearchTechnologies) was spiked into the sample and the fluorescence monitoredover 20 minutes. Additional measurements were taken at 25, 50, 75, and200 nM concentrations of mRNA. This same procedure was repeated for theDNAzyme-NAN (10 nM). Note, as it was determined that there areapproximately 2 DNAzymes per surfactant molecule in a given DNz-NANassembly, the value of 20 nM DNAzyme was utilized for the ([ET]) in theKcat calculation. Kcat=Vmax/[ET]. For the free DNAzyme calculation, 10nM was used for ([ET]). All non-linear fits for assigning Vmax valueswere determined using Kaleidagraph 4.5 graphing software.

Discussion:

Ester-crosslinked NANs were treated with T4 DNA ligase to provideDNAzyme covalently assembled at the nanocapsules. The DNAzyme wassynthesized using automated DNA synthesis and ligated to a DNA anchor atthe NANs surface. Characterization of all DNAzyme-NAN materials used inthese studies consisted of dynamic light scattering (DLS) (FIG. 13A) andagarose gel electrophoresis shift assays (FIG. 13C). The average size ofthe micellular core was shown to increase from 23.4±5 nm to 63.5±10 nmpost DNA attachment, indicative of addition of the DNAzyme to thesurface of the DNA-functionalized nanocapsule. This can be determinedbased on the fully extended length calculation for the unfolded DNAzyme,a 33mer sequence.

To determine the total number of DNAzymes per surfactant, the DNAzymewas modified with a 5′ terminal TYE-563 dye, and ligated to the NANs.Unligated dye labeled DNAzymes were then removed by size exclusionchromatography. Using a standard curve of the emission from the TYE-563dye labeled DNAzyme, the remaining fluorescence of the NANs post DNAzymeligation could be determined and used for subsequent concentrationcalculations for treatment with cells. Notably, the average number ofDNAzymes per surfactant came out to be 2.3±0.2. This suggests a highlyefficient stepwise construction of DNA ligands on the surface of thenanocapsule. Attachment of the oligonucleotides occurs in two steps,starting with the photocatalyzed attachment of the 5′ thiolated DNAanchor to the terminal alkynes of the crosslinked micelle's surfactantmolecules, followed by enzymatic ligation of the DNAzymes to the DNAanchor molecules. Once assembled, comparative DNAzyme cleavage kineticswere investigated using a fluorophore and quencher labeled GATA-3 mRNAtruncate (19mer) described previously. Upon cleavage of the labeled mRNAtarget, a quenched dye becomes fluorescent and can be monitored as afunction of substrate concentration over time. DNAzyme concentration washeld constant (10 nM) and the concentration of mRNA truncate varied from10-200 nM. The rate of cleavage was monitored over the course of 20minutes. Changes in fluorescence were plotted and the initial rise ateach concentration was fit to determine an observed rate of cleavage pernanomolar amount of substrate (FIG. 14). The results indicate that theDNAzyme follows a classical Michaelis-Menten type model of saturationkinetics, with the rate of cleavage saturating at higher mRNA substratelevels (FIG. 14). The relative rates of cleavage by the DNAzyme versusthe DNz-NAN are shown to be roughly comparable. The DNAzyme wasdetermined to have a K_(m) of 59 nM and a K_(cat)/K_(m) of 1.1×10⁶ M⁻¹min⁻¹, whereas the DNz-NAN had a K_(m) of 32 nM, and a K_(cat)/K_(m) of7.5×10⁵M⁻¹min⁻¹. This indicates that the immobilization of the DNAzymeon the NANs surface by its 5′ end enables a rapid rate of cleavage but aslightly decreased activity overall, likely due to the crowded surfaceon the nanocapsule as a result of neighboring DNAzymes. However, theconstructs differ in efficiency by a factor of roughly 1.5, indicatingthat when tethered, the DNAzyme does not suffer a significant decreasein activity as has been observed in other colloidal DNAzyme systems.

Targeted cleavage is only possible if the DNz-NAN can disassemble andthe DNz-surfactants can escape the endosomal compartments of the cellpost endocytosis. To study the suitability of the DNz-NAN for cellularuptake and stability of its DNAzyme ligands in the cell, the DNz-NANswere first incubated in 10% fetal bovine serum to mimic exposure tocellular nucleases. Using a PCR assay that was developed to determinethe presence of full length DNAzyme, it was found that after exposure to10% FBS for 1 hr at 37° C., full length DNAzyme could still be observedon the DNz-NAN surface (FIG. 13D). The DNAzyme was also synthesized witha terminal dye (TYE-665) and ligated to the NAN for cellular tracking. 1μM Dye-DNz-NANs were incubated with MCF-7 cells for 4 hr and monitoredfor evidence of uptake using confocal microscopy (FIGS. 15A and 15B).Next, gene knockdown by the DNz-NAN was compared to that of siRNA andfree DNAzymes (both delivered using lipofectamine-2000), wherein theDNz-NANs performed comparably, resulting in 60% knockdown of GATA-3(FIG. 15C). Furthermore, it was found that the DNz-NAN showed apersistence of knockdown that continued for over 12 hrs. By 48 hr, themRNA levels of GATA-3 showed evidence of recovery, although still at 60%relative to the untreated cells (FIG. 15D).

A synthetic lipoplex system was designed to help monitor mRNA cleavageacross a lipid bilayer (FIG. 16A). This construct was built using a goldnanoparticle (Au NP) as it can support a simplified lipid bilayer system(FIG. 16B). At the Au NPs surface, both a thiolated DNA molecule and athiolated PEG-DSPE molecule were assembled (1:2 ratio). The DNA at thesurface serves as an anchoring site for the annealing of the 19mer mRNAtruncate. For this assay, the same mRNA truncate that was utilized inthe kinetics assay was used. Therefore, unless cleaved, the moleculeshould have limited fluorescence as it has both the BHQ quencher and FAMmodifications. This construct was further encapsulated with DOPE, toform a bilayer around the AuNPs, a procedure previously disclosed (Shenet al. Nanoscale, 2016, 8, 14821-14835.) These particles were thensubjected to either the free DNAzyme or DNAzyme-surfactants, and theextent of mRNA cleavage evaluated as a function of increases observed inoverall fluorescence. To mimic more closely the DNz-surfactant thatwould results from the DNz-NANs post degradation by endosomal esterases,a modified surfactant which presented only two alkynes was synthesized,attached the DNA anchor to it, and ligated DNAzymes to the anchor toform a DNAzyme-surfactant conjugate. The results indicate that thehydrophobically modified DNAzyme can access the mRNA truncate and cleaveit, whereas the DNAzyme alone appears to show limited change in signal,likely do to its inability to access the mRNA truncate (FIG. 16E).Changes in fluorescence by the free DNAzyme compared to changes influorescence by background salts (control sample) shows an overallminimal change in mRNA fluorescence, also indicating limited access tothe mRNA target. All conditions had equal concentrations of DNAzyme.

Taken together, these results show that the multifunctionalnanoparticles of the disclosure are a successful delivery system forintracellular nucleic acid delivery, and an effective gene knockdownstrategy, which avoids common drawbacks such as the use of traditionalcationic transfection agents and further chemical modifications. Themultifunctional nanoparticles of the disclosure provide an effectivenucleic acid delivery into the cell and the hydrophobic surfactantmodification of the DNAzyme enables uptake and access to the mRNAtarget. Lastly, the delivery of the DNAzyme-NANs of the disclosureresulted in specific and persistent gene knockdown of a target gene,GATA-3, for several hours. In certain embodiments, the multifunctionalnanoparticles of the disclosure may be used for co-delivery ofhydrophobic drugs and oligonucleotides.

Example 6

Ester-crosslinked NANs of Example 1 were loaded with sudan II andincubated with HeLa cells. Compared to cells treated with NANs withoutSudan II dye, fluorescence was observed in HeLa cells treated at 0.5 μMNANs. Next, MMT was used to evaluate cell viability. As illustrated inFIG. 17, unloaded NANs maintained ˜80% cell viability up to 2 μM, abovecell treatment concentrations. When loaded with ABT-737 (a smallmolecule inhibitor of Bcl-2 family proteins), significant cell death isobserved.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

1. A multifunctional nanoparticle comprising one or more of nucleic acidligands covalently attached to a particle comprising non-polymericamphiphiles, wherein hydrophobic groups of the amphiphiles are arrangedtoward the particle interior, and wherein hydrophilic groups of theamphiphiles are at the particle surface and are crosslinked through atriazole, thioether, or alkenyl sulfide group with one or more peptidelinkers cleavable by one or more intracellular or extracellular releaseagents.
 2. The multifunctional nanoparticle of claim 1, wherein thehydrophobic groups of the amphiphile comprise C₆-C₂₂ alkyl, C₆-C₂₂alkenyl, or C₆-C₂₂ alkynyl group
 3. The multifunctional nanoparticle ofclaim 1, wherein the hydrophilic groups of the amphiphile comprise anammonium group.
 4. The multifunctional nanoparticle of claim 1, whereinthe linker is cleavable by an enzyme.
 5. (canceled)
 6. Themultifunctional nanoparticle of claim 1, wherein the peptide linkercomprises GPLGLAGGERDG (SEQ ID NO:10), GFLG (SEQ ID NO:11), GPMGIAGQ(SEQ ID NO:12), Phe-Leu, Val-Ala, Val-Cit, Val-Lys, Val-Arg, or Phe-Lys.7. (canceled)
 8. (canceled)
 9. The multifunctional nanoparticle of claim1, wherein the linker comprises a disulfide group.
 10. Themultifunctional nanoparticle of claim 1, wherein the nucleic acidligands are capable of selectively binding to a cell surface antigen.11. The multifunctional nanoparticle of claim 1, wherein the nucleicacid ligand is capable of gene regulation, and wherein the nucleic acidis siRNA, DNAzyme, ribozyme, microRNA, or other therapeuticoligonucleotide.
 12. The multifunctional nanoparticle of claim 1,wherein the nucleic acid ligand is capable of selectively binding to aprotein, wherein the protein is selected from the group consisting oftumor-markers, integrins, cell surface receptors, transmembraneproteins, ion channels, membrane transport protein, enzymes, antibodies,and chimeric proteins.
 13. The multifunctional nanoparticle of claim 1,wherein the nucleic acid ligand is capable of selectively binding to acarbohydrate, wherein the carbohydrate is selected from the groupconsisting of glycoproteins, sugar residues, and glycocalyx.
 14. Themultifunctional nanoparticle of claim 1, wherein the nucleic acid ligandis capable of selectively binding DNA, RNA, modified DNA, modified RNA,DNAzymes, ribozymes, mRNA, siRNA, microRNA, shRNA, and combinationsthereof.
 15. The multifunctional nanoparticle of claim 1, wherein thenon-polymeric amphiphiles are derived from

wherein X is halogen.
 16. A conjugate comprising the multifunctionalnanoparticle of claim 1 and at least one therapeutic agent or diagnosticagent, wherein the multifunctional nanoparticle encapsulates thetherapeutic agent or diagnostic agent.
 17. The conjugate of claim 16,wherein the conjugate comprises a therapeutic agent which is ahydrophobic small molecule drug selected from group consisting of ananti-cancer agent, an antibiotic, an antiviral, an antiparasitic agent,an anticoagulant, an analgesic agent, an anesthetic agent, an ionchannel potentiator, an ion channel inhibitor, an anti-inflammatory, ametallodrug, and any combination thereof.
 18. The conjugate of claim 16,wherein the conjugate comprises a diagnostic agent, which is afluorophore, a radiolabeled nucleotide, a radioisotope, biotin,tocopherol, cholesterol, a steroid, or a electron dense tag and a metalchelator.
 19. A method of treating a disease or disorder, comprisingadministering to a subject in need thereof an effective amount of theconjugate of claim 16, wherein the linker is cleavable by one or moreintracellular or extracellular release agent present in the subject,thus releasing the therapeutic agent or diagnostic agent.
 20. The methodof claim 19, wherein the release agent is a lysosome agent, endosomeagent, caveolae agent, or an enzyme expressed by tumor cells.